Sex determination is a fundamental developmental decision in which the bipotential gonad is directed into specific pathways of male or female development. Despite the central importance of this process, there is still much to learn about its underlying molecular mechanisms. Our long term goal is to discover and characterize the gene regulatory network responsible for this central developmental switch in vertebrates. We have three specific aims: 1) to use the extensive genomic resources we have developed for cichlid fishes to complete the genetic mapping of male heterogametic (XY) sex determining loci on LGs 1 and 7, and female heterogametic (WZ) systems on LGs 3 and 5. Characterization of the critical map interval for each locus is the first step in identifying the top-level regulatory genes in each species. 2) to use quantitative PCR and in situ hybridization to quantify the normal pattern of gene expression in the developing gonads of our model system, the Nile tilapia. We will then examine the spatial and temporal expression of candidate sex-determining genes within this framework. 3) to quantify patterns of gene expression in response to both natural perturbations (e.g. the natural differences among the four sex-determining systems) and the experimental upregulation of gene expression by means of transgenesis. This combination of natural and experimental manipulations will allow us to probe the structure of the regulatory network in detail. Together, these experiments will provide insights about the gene networks regulating vertebrate sex determination. How many genes can control the sexual fate of vertebrates? Do the nodes which become top-level regulators of sex determination have unique characteristics? Does the sex- determining pathway have a modular organization, and if so, what is the relationship of the top-level regulators to these modules? This work will also facilitate the identification of genes which contribute to aberrant sexual development and gonadal tumors in humans. Project Narrative: Defects in gonadal specification and differentiation are among the most common developmental defects, resulting in partial sex reversal, infertility and various cancers. This project will characterize portions of the complex genetic control network responsible for sex determination and gonadal development that are shared among vertebrate species. This knowledge will contribute to the discovery of new therapies for these defects in humans.